Peripheral arterial disease (PAD) causes reduced blood flow in distal limb tissue. In some patients, critical limb ischemia will develop if the perfusion deficit is not effectively treated. The restoration of blood flow via the arteriogenic growth of collateral arteries bypassing the occlusion(s) may eventually represent a therapeutic option for these patients. However, our still incomplete understanding of arteriogenesis has limited our ability to translate therapeutic arteriogenesis to the clinic. In this proposal, we aim to better understand the role that shear stress-mediated changes in endothelial cell DNA methyltransferase (cytosine-5) 1 (DNMT1) expression have on the capacity of collateral vessels to enlarge through arteriogenesis. This proposal follows logically from our recent discovery that, in the mouse femoral artery ligation (FAL) model, collateral segments exposed to reversed flow exhibit remarkably amplified and sustained arteriogenesis. In pilot studies, we determined that endothelial cells (ECs) exposed to a biomimetic non- reversed flow (i.e. mild arteriogenesis) waveform exhibit genome-wide DNA hypermethylation and enhanced DNA methyltransferase 1 (DNMT1) expression when compared to ECs exposed to a biomimetic reversed flow waveform (i.e. amplified arteriogenesis). In Aim 1, we will determine whether pro-arteriogenic EC responses to hemodynamic stimuli (i.e. elevated shear stress magnitude +/- reversed flow direction) are modulated by DNMT1 expression and subsequent DNA methylation levels. We will knockdown and overexpress DNMT1 in ECs exposed to these waveforms and assay for pro-arteriogenic EC responses. In Aim 2, we will then determine whether enhanced EC DNMT1 expression, elicited by exposure to elevated shear stress, compromises arteriogenic capacity by increasing shear stress ?set-point?. This aim is motivated by studies showing that collateral segments exposed to increased flow without reversed flow direction exhibit insufficient arteriogenesis, such that their lumens remain too narrow to fully restore shear stress to its original value. However, inhibiting DNA methylation with 5-AZA allows these segments to enlarge and restore shear stress. This leads us to the hypothesis that the arteriogenic capacity of collateral arteries exposed to increased shear- stress is diminished by increased EC DNMT1 expression and subsequent DNA hypermethylation. Here, we will first generate inducible EC-specific DNMT1 knockout mice through breeding. The DNMT1 gene will be excised from ECs, either at the time of FAL surgery or 2 weeks after FAL, when collateral diameters have already reached their steady state. Arteriogenesis will be compared to control mice, as well as within FAL operated hindlimbs by comparing reversed and non-reversed flow collateral segments. Ultimately, if our hypothesis is verified, we believe it may have substantial clinical impact because it would reveal that EC DNA hypermethylation could be a major limiting factor in the ability of endogenous and/or therapeutic arteriogenesis to restore distal perfusion in the presence of arterial occlusion(s).